ES2534652T3 - Antibodies against glycoprotein VI for the recombinant platelet collagen receptor - Google Patents

Abstract

Polyclonal anti-GPVI antiserum, anti-GPVI Fab obtainable from said anti-GPVI antiserum, or F (ab ') 2 anti-GPVI obtainable from said anti-GPVI antiserum, wherein GPVI has the sequence of SEQ ID NO: 3.

Description

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DESCRIPTION

Antibodies against glycoprotein VI for the recombinant platelet collagen receptor

The invention relates to glycoprotein VI (GPVI), its isolation, purification, and methods for recombinant production. Especially, the invention relates to the use of GPVI, preferably recombinant GPVI, in the

5 treatment of disorders and pathological events directly or indirectly correlated with blood coagulation disorders, such as thrombotic and cardiovascular diseases. The extracellular recombinant protein can also be used to establish screening assays to find potential inhibitors of membrane-bound GPVI in order to inhibit the interaction of thrombocytes and collagen.

Glycoprotein VI is a 62/65 kDa platelet membrane glycoprotein (not reduced / reduced

10 respectively), which forms a complex together with the common subunit Fc. The GPVI subunit contains the collagen binding site, and the Fc subunit is responsible for signaling. The complex forms one of the main collagen receptors on the platelet surface, critical for platelet activation in response to collagen. The collagen recognition sequence consists of sequences of (GlyProHyp) n ’. Patients from Japan who have a genetic deficiency of GPVI are known. They have mild bleeding problems, and

15 their platelets respond only weakly to collagen, presumably through other receptors. Much has been learned about signaling cascades that originate in GPVI, which closely resemble those of immune receptors, including T-lymphocyte receptors, B-cell receptors and natural killer cell receptors. These cascades involve the src family of tyrosine kinases such as Fyn and Lyn, as well as p72SYK and many other tyrosine kinases and phosphatases, and adaptive proteins such as LAT. A main target of these

20 cascades is the activation of the Cy2 phospholipase that unfolds phospholipids to give the second messengers diacylglycerol and IP3. GPVI is thought to be involved in the activation of platelet integrin B2B1, which has a major role in the adhesion of platelets to the wall of damaged vessels. Mice with the Fcy "genetically removed" subunit have platelets that still show responses to collagen, implying that the resting state of 2B1 can also be regulated by the GPVI / Fcy complex.

25 The GPVI of the platelet collagen receptor is closely related to the natural killer activating receptors of the p58KAR family.

Adhesion and activation of circulating resting platelets at a vascular lesion site is the first stage in a process that leads to the formation of a thrombus, which becomes a hemostatic plug. Collagen is one of the main components of the vessel wall, responsible for the activation of platelets. There are 30 many types of collagen, and seven of these are found in the subendothelial layers. Several different receptors for collagen have been identified in platelets, but the main ones are now considered to be integr2B1 integrin and non-integrin GPVI. Although 2B1 is well characterized and both subunits were cloned and sequenced several years ago, the structure of the GPVI has remained difficult to find out, although several traits have been identified. It was determined, approximately twenty years ago, that GPVI is an important platelet glycoprotein with a molecular mass in the range of 60-65 kDa and an acidic pl. Its role as a putative collagen receptor was established after the identification of a patient in Japan with a mild bleeding disorder whose platelets showed a specific defect in collagen response and lacked this receptor. This patient had also developed autoantibodies for the deficient receptor, and these were used to further characterize the molecule. More recently, it was established that GPVI is non-covalently associated with the common Fcy subunit, which acts as the signaling part of the complex. It was also shown that the recognition sequence in collagen for GPVI is a Gly-Pro-Hyp triplet repeated within the triple helical structure of collagen, and that synthetic peptides based on this structure could be used as specific agonists targeting GPVI . It was shown that the GPVI / Fcy complex signals into the platelets through a mechanism similar to an immune receptor, which involves the activation of 45 p72SYK and that leads through a cascade of kinase / phosphatase / protein interactions adapting to the activation of PLCy2 and therefore to the release of granules and platelet aggregation. An additional stage in the characterization of this molecule was the demonstration that the type C snake lectul, convulxin, of the Tropical Rattlesnake, Crotalus durissus terrificus, was able to activate platelets by grouping GPVI through a multimeric interaction. It was shown that convulxin binds specifically to GPVI, providing a

50 method for purification of this receptor along with established approaches.

Sugiyama et al. (Blood 69 (1987) 1712-1720) describe a new platelet aggregating factor. This factor has been found in a patient with autoimmune thrombocytopenia. The factor is the patient's IgG or its Fab or F (ab) 2 fragments. F (ab) 2 fragments induced irreversible aggregation in normal platelet rich plasma. In addition, Fab fragments specifically inhibited the aggregation induced by collagen and by the patient's IgG. He

The main component of an immunoprecipitate obtained with the patient's IgG from radiolabeled membrane proteins of normal platelet extract had a molecular weight of 62 kDa.

Sugiyama et al. (International Journal of Hematology 58 (1993) 99-104) examined the functional role of P62, the antigen recognized by the IgG Fab fragment isolated from the patient with autoimmune thrombocytopenia. It is described that the patient's platelet showed normal adhesion to collagen, and that the Fab fragment inhibited the

60 normal platelet adhesion to collagen in a dose-dependent manner.

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Ichinohe et al. (The Journal of Biological Chemistry 270 (1995) 28029-28036) examined the role of GPVI protein using F (ab) 2 fragments of the IgG of the patient with autoimmune thrombocytopenia. The GPVI coupling is coupled to the activation of c-Src and Syk, insensitive to cAMP, accompanied by tyrosine phosphorylation of numerous substrates, including C-2 phospholipase, in a manner similar to collagen stimulation.

5 Gibbins et al. (FEBS Letters 413 (1997) 255-259) describe that GPVI is the platelet collagen receptor that couples the stimulation of platelets by collagen to the tyrosine phosphorylation of the  chain of the Fc receptor. The authors use anti-GPVI F (ab) 2 fragments of the IgG of the patient with autoimmune thrombocytopenia.

Jandrot-Perrus et al. (The Journal of Biological Chemistry 272 (1997) 27035-27041) describe the adhesion and activation of human platelets induced by convulxin. Adhesion and activation involves GPVI and integrin

10 alpha2beta1. Platelet exocytosis and aggregation induced by convulxin were inhibited by IgG Fab fragments of the patient with autoimmune thrombocytopenia.

Ezumi et al. (The Journal of Experimental Medicine 188 (1998) 267-276) investigated the involvement of the Src family in proximal signals through the GPVI-FcR complex using convulxin and F (ab) 2 anti-GPVI fragments of the patient's IgG with autoimmune thrombocytopenia. The results indicated that Src's family plays

15 a critical role in platelet activation via the GPVI-FcRy collagen receptor complex.

Heemskerk et al. (Thrombosis and Haemostasis, Schattauer GmbH 81 (1999) 782-792) describe the role of GPVI and integrin alfa2beta1 in the procoagulant response of individual collagen adherent platelets. The authors use anti-GPVI IgG and the corresponding Fab fragments of the IgG of the patient with autoimmune thrombocytopenia.

20 Ishibashi et al. (International Journal of Hematoloy 62 (1995) 107-115) identified the intercellular adhesion molecule 2 (ICAM-2) using the antibody of the patient with autoimmune thrombocytopenia in an improved immunoprecipitation technique. The ICAM-2 molecule has a molecular weight of about 62 kDa.

EP 0,896,002 refers to chimeric proteins consisting of an integrin and an immunoglobulin, their heterodimer complexes, a method of production thereof and their applications as drugs and reagents. In addition, EP 0.896.002 refers to the medical application of the chimeric integrin-immunoglobulin protein as a platelet substitute. Integrins are molecules that belong to the integrin superfamily. Alpha chains include 15 alpha chains, among others, alpha1. The beta chains include eight beta chains, among others, beta1. It was found that a heterodimer complex of chimeric protein integrin alfa2beta1-immunoglobulin has the ability to bind to the extracellular matrix. Therefore, it can be used as

Therapeutic or preventive agent against congenital and acquired hemorrhagic tendency due to platelet abnormality, and also widely as a substitute for platelet transfusion. In addition, the chimeric protein integrin alpha2beta1-immunoglobulin heterodimer complex may be a therapeutic or preventive agent for disease conditions in which vascular endothelial cell disorder is a problem.

Therefore, it is clear from the prior art that GPVI seems to be a very interesting compound in many 35 therapeutic fields, especially concerning applications that are related, generally speaking, directly

or indirectly, with blood clotting events that depend on the collagen-platelet interaction. It was, therefore, a goal of the present invention to provide GPVI in a recombinant form and to show its efficacy as a direct therapeutic target or as a tool for screening short compounds, especially chemically synthesized or synthesizable compounds that have the ability to inhibit or block the interaction

40 natural platelet-collagen.

It was another goal of the present invention to provide anti-GPVI antibodies.

In view of this, the present invention relates to a polyclonal anti-GPVI antiserum, an anti-GPVI Fab obtainable from said anti-GPVI antiserum, or an anti-GPVI F (ab ') 2 obtainable from said anti-GPVI antiserum, in which GPVI has the sequence of SEQ ID NO: 3.

The present invention also relates to a polyclonal anti-GPVI antiserum, an anti-GPVI Fab obtainable from said anti-GPVI antiserum, or an anti-GPVI F (ab ') 2 obtainable from said anti- serum antiserum GPVI as described above, in which GPVI can be obtained from platelets using wheat germ agglutinin and convulxin and applying gel electrophoresis on polyacrylamide of about 8.5%.

The present invention further relates to a polyclonal anti-GPVI antiserum, an anti-GPVI Fab obtainable from

50 of said anti-GPVI antiserum, or an anti-GPVI F (ab ’) 2 obtainable from said anti-GPVI antiserum as described above, wherein the polyclonal anti-GPVI antiserum is generated in rabbits.

In addition, the present invention relates to an anti-GPVI Fab fragment of humanized mouse monoclonal antibodies against GPVI, in which GPVI has the sequence of SEQ ID NO: 3.

The present invention also relates to monoclonal or polyclonal antibodies that recognize the N55 terminal region of GPVI, in which GPVI has the sequence of SEQ ID NO: 3.

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The invention describes portions or fragments of the GPVI protein that have maintained their biological activity, which is the binding to collagen.

The invention was successful in purifying adequate amounts of GPVI for preliminary characterization and for peptide sequencing. The sequences were used to design primers for PCR to identify a

5 positive sequence in a DNA library. This DNA sequence was then used as a probe to isolate an almost complete cDNA sequence from the library and the missing 5 ′ sequence was obtained using a RACE method from a platelet cDNA library.

The invention also succeeded in showing the use of recombinant GPVI as a therapeutically applicable compound that is capable, when administered to a patient with, for example, damaged blood vessels, of binding to collagen, thus preventing thrombocytes carrying GPVI bound to the membrane bind to said collagen. The recombinant soluble extracellular domain of GPVI contains the collagen binding site and may prevent platelet activation by collagen. It could, therefore, be applicable to the treatment of disease states that involve increased activation of platelets with collagen, such as atherosclerotic plaque rupture, in diseases such as unstable angina or during surgical treatment, such as surgery. Percutaneous Transluminal Coronary Angioplasty (PTCA), in which the arteries are reopened by inflating a balloon catheter causing considerable damage to the vascular wall and much platelet activation, and often resulting in the vessel closing again later. The advantage of recombinant GPVI fragments compared to current treatment methods is that they act at an earlier stage preventing or reducing platelet activation rather than suppressing events after platelet activation, such as aggregation by antagonists of GPIIb-IIIa. Thus, smaller amounts of the contents of the platelet granules are released, including growth factors and chemokines, which are involved not only in wound repair but in the remodeling of the vascular wall by smooth muscle migration and in the attraction of phagocytic cells such as monocytes, which are known to contribute to atherosclerosis. The Fab fragment of humanized mouse monoclonal antibodies against GPVI could be used

25 with a similar effect to block GPVI on the platelet surface with similar applications as the previous ones.

Recombinant GPVI as described herein can also be used in a collagen binding assay to screen small molecules (in combinatorial libraries for example) capable of inhibiting this interaction and that can be used to develop therapeutic compounds that are inhibitors of collagen interaction. -platelet. By proper derivatization, these compounds are made available orally. Again, the main objective is to prepare compounds that reduce GPVI-collagen interactions and therefore the activation of platelets in situations where platelets come into contact with collagen. The screening technology as such used in this invention is well established in the prior art. Through such screening assays, the invention allows to find and develop new targets that can inhibit the membrane-bound natural GPVI on the surface of the

35 thrombocytes as a collagen antagonist. Such targets, which can be short chemical molecules, can then be the basis for subsequent inventions.

Another main application of GPVI and reagents that recognize specific domains of GPVI is as markers of platelet age and functionality. It is generally thought that young platelets are more active and functional than older ones. Young platelets bind to and are activated by the type 40 C lectin of convulxin snake venom, which is specific for GPVI, and as they age they decrease both the binding and the degree of activation. This may be due to either proteolytic or conformational changes in the GPVI

or its association with Fcy due to platelet activation or circulation damage. This can be a useful parameter to measure the age profile and function of platelets in patients, as well as in normal people, during medical checks. The age profile of platelets changes in many diseases that affect the bone marrow or immune system, and could be an important diagnostic criterion if better methods were available for their determination. For example, patients with diseases that involve increased platelet turnover will show more young platelets, while patients undergoing chemotherapy or radiation treatment will show a population that is continuously aging. In this way, such an age profile can be used for precise monitoring of the treatment. In a normal healthy population, very little is known about the distribution of the age profile and its role as a predictor of health changes. Thiazole orange is currently used to detect young cross-linked platelets containing mRNA. This mRNA decomposes soon, restricting the method only to younger platelets. Reagents that could be used in such an assay would include snake venom proteins specific for GPVI, such as convulxin, or monoclonal or polyclonal antibodies that recognize the N-terminal region of GPVI, or monoclonal antibodies that recognize new sites or conformations exposed by proteolysis of the N-terminal domain or specific conformations present either in the intact molecule and not in the aged or vice versa. These reagents would be labeled with a fluorescent label, or together with a second antibody or fluorescent labeled affinity reagent, and would be used in flow cytometry to measure platelet binding profile. At a later alternative stage, less laborious measurement techniques could be adopted based on a measurement

60 automated platelet profiles. Using cell sorting methods with flow cytometry or magnetic beads, it should be possible to isolate young and old platelets to examine the factors involved in removing old platelets from the circulation.

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The present invention describes a DNA encoding Glycoprotein VI or ss biologically active fragments, especially the sequence of Fig. 2.

The present invention further describes a DNA encoding Glycoprotein VI comprising the amino acid sequence of Fig. 1.

The present invention describes a pharmaceutical composition comprising recombinant GPVI together with a pharmaceutically acceptable diluent, vehicle or excipient thereof, and its use for the manufacture of a medicament in the therapeutic field of thrombotic and cardiovascular events and disorders related to platelet interactions. collagen

The present invention describes the use of recombinant GPVI in a screening tool to detect

10 specific inhibitors of platelet-collagen interactions. Possible indications and medical applications, respectively, are, for example, unstable angina pectoris, PTCA, the use of stents in this field, operations in coronary vessels, general operations in blood vessels, operations that may damage larger blood vessels, such as hip joint operations. In addition, all indications referring to thromboembolic events caused by disorders of the body are included.

15 interaction between the vessel wall and the coagulation system with a high risk of thrombus formation and vessel blockage.

As indicated above, GPVI protein and fragments thereof are suitable as pharmaceutically effective compounds in pharmaceutical compositions and combinations.

Pharmaceutical formulations may optionally comprise additional active ingredients, such as

20 anticoagulants such as hirudin or heparin, or thrombolytic agents such as plasminogen activator or hementin.

The new protein, and its biological active fragments, respectively, can form pharmaceutically acceptable salts with any non-toxic organic or inorganic acid. Inorganic acids are, for example, hydrochloric, hydrobromic, sulfuric or phosphoric acid and acidic metal salts, such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Examples for organic acids are mono-, di-and tricarboxylic acids such as acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxyloleic, benzoic, hydroxybenzoic, phenylacetic , cinnamic, salicylic, and sulfonic acids such as methanesulfonic acid. Salts of the carboxy terminal amino acid residue include non-toxic salts of carboxylic acids formed with any suitable inorganic or organic bases. These salts include, for example, alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, light metals of Group IIIA, including aluminum, and primary, secondary and tertiary organic amines such as trialkylamines, including triethylamine, procaine, dibenzylamine, 1-ethenamine, N, N'-dibenzylethylenediamine, dihydroabyethylamine and N-alkylpiperidine. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid or liquid filler, diluent or encapsulating material that does not react.

35 adversely with the active compound or with the patient. Suitable vehicles, preferably liquids, such as sterile water, saline solution, aqueous dextrose, sugar solutions, ethanol, glycols and oils, including those of petroleum, animal, vegetable or synthetic origin, for example peanut, soybean oil and mineral oil.

The formulations can be administered as unit doses containing the excipients, diluents, adjuvants

40 and pharmaceutically acceptable, non-toxic, conventional vehicles, which are typical for parenteral administration.

The term "parenteral" includes herein subcutaneous, intravenous, intraarticular and intratracheal injection and infusion techniques. Other administrations, such as oral administration and topical application are also suitable. Parenteral compositions and combinations are more preferably administered by route.

IV, either in a bolus form or as a constant fusion, according to known procedures. Tablets and capsules for oral administration contain conventional excipients such as binding agents, fillers, diluents, tabletting agents, lubricants, disintegrants, and wetting agents. The tablets may be coated according to methods well known in the art.

Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions,

50 emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, emulsifying agents, non-aqueous vehicles and preservatives.

Topical applications may be in the form of aqueous or oily suspensions, solutions, emulsions, jellies or preferably emulsion ointments.

The unit doses may contain the daily required amounts of the protein according to the invention, or submultiples thereof to compose the desired dose. The therapeutically acceptable optimal dose and the frequency of the dose for a given patient (mammals, including humans) depend on a variety of factors, such as the activity of the specific active material employed, age, body weight, general health, sex, diet, time and route of administration, speed of clearance, the object of treatment, that is, therapy or prophylaxis, and the nature of the thrombotic disease to be treated, antiplatelet or anticoagulant activity.

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Therefore, in compositions and combinations useful as anticoagulants in a treated patient (in vivo), a

The pharmaceutically effective daily dose of the peptides of this invention is between about 0.01 and 100 mg / kg body weight, preferably between 0.1 and 10 mg / kg body weight. Depending on the form of application, a single dose may contain between 0.5 and 10 mg of the thrombin inhibitor. To achieve an anticoagulant effect in extracorporeal blood, a pharmaceutically effective amount of the inventive peptides is between 0.2 and 150 mg / l, preferably between 1 mg and 20 mg / l of extracorporeal blood.

10 Detailed description of the invention

Two sequences of 7 amino acids were chosen that showed minimal degeneracy in the genetic code for the synthesis of DNA primers, in order to amplify part of the GPVI cDNA by PCR. As the location of both peptides in the protein was totally unknown, for each of them, two degenerate primers, one felt and one antisense, were prepared. These primers were used to amplify a bone marrow library

15 human. The combination of the 5’TYA THC CNG CNA TGA ARMG 3 ’sense primer encoding the PAMKRSL sequence with the antisense 5’TTR TAN RNA GCR AAY TGR TC 3’ corresponding to DQFALYK amplified a 221 bp DNA fragment. In addition to the selected peptides, the amplified DNA encoded the LysC / AspN DQLELVATGVFAKPSLSAQPGPAVSS peptide, which clearly binds the sequence to the cDNA for GPVI.

Screening of 600,000 pfu from a bone marrow library with this 221 bp DNA fragment produced 4 pfu

20 positive Three had 1350 bp inserts either cut by the restriction enzyme Sal I or by EcoR I and belonged to the IgG superfamily. The fourth had a 4.6 kb insert by digestion with Salt I and gave two 2300 bp and 1300 bp fragments respectively when treated by EcoR I. Its DNA contained the sequence for the 10 peptides derived from amino acid sequencing of the GPVI but stopped near the amino terminal. No starting methionine or leader sequence could be found, but more than 2000 bp of non-DNA was present.

25 previously sequenced reading frame ending in an Alu sequence. The RACE experiment of the 5 ′ end was completed in platelet poly A RNA with primers located in a part of the GPVI sequence that had been corroborated by that of the peptides. A 318 bp fragment that includes 70 new bp and x bp in the fourth pfu sequence in bp 1987 corresponding to 14 amino acids that include the first methionine was found before falling into the established GPVI sequence. In this way, a cDNA could be sequenced that

30 contained a total of 1249 bp and encodes a protein with an open reading frame that includes the leader sequence with 339 amino acids.

A cDNA encoding platelet GPVI was cloned and sequenced from a human bone marrow cDNA library using RACE with platelet mRNA to deliver the missing 5 ′ sequence. The 1017 bp open reading frame encodes 339 amino acids and a 3 ’untranslated region. Hydrophobicity analysis of the 35 amino acid sequence revealed the presence of two putative transmembrane domains, a putative 23 amino acid signal sequence, and a 19 amino acid domain between residues 247 and 265 of the mature protein. The sequence and its amino acid translation are shown in Fig. 2 and Fig. 1. A comparison with the amino acid sequence of the most similar molecules found in a GenBank search clearly reveals that it belongs to the immunoglobulin superfamily, and The extracellular domain contains two Ig C2 domain loops formed by two disulfide bridges. It is a protein molecule that crosses the class one membrane, with the term N on the outside and crosses the membrane once. The most closely related molecules belong to the class of natural killer receptors, which contains both inhibitor and activator types. GPVI clearly belongs to the activating subclass, not only through its function but also because, unlike the inhibitory class, it does not contain ITIM sequences in its cytoplasmic domain. It also does not contain any tyrosine residues that might be involved in phosphorylation. There are some threonine and serine residues in this domain, but they do not match any criteria for kinase consensus sequences. Like the NK receptor activator class, GPVI contains an arginine residue as the third amino acid in the membrane-crossing domain, which is involved in the formation of the complex with the Fcy subunit. The cytoplasmic domain contains 51 amino acids, showing only a small similarity (in the region just below the membrane) to the 50 cytoplasmic domains of other members of this family. This suggests that this domain in GPVI can be associated with different types of cytoplasmic molecule than the other family members. GPVI contains only a single putative N-glycosylation site in Asn69. The domain just above the membrane before the beta sheets of the Ig domain begin, however, is rich in threonine and serine residues, which could provide O-glycosylation sites such as those found in the GPIb and GPV. The function

The main part of this O-glycosylation seems to be to present the receptor structures well extended from the platelet surface to facilitate interactions with their bulky ligands. Since GPVI was previously established as a sialoglycoprotein, the difference in molecular mass between the theoretical amino acid mass (37 kDa) and the mass determined by gel electrophoresis (reduced 65 kDa) should be due to this glycosylation.

The structure of natural killer receptors of the two types of domain has been established by studies

X-ray crystallographic, and it was shown that the two Ig domains form an acute angle, the receptor site being found for HLA antigens carrying peptides outside the elbow. A direct comparison of the

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The structure of the HLA peptide binding site with that of the collagen immediately suggests that these receptors have a common origin, because the multiple alpha-helical structures of the HLA binding site and the peptide it contains closely resemble the structure of triple helix of collagen. In addition, GPVI contains multiple PGX sequences, which could mimic an additional collagen strand in interaction with a collagen fiber. 5 It has been postulated that natural killer receptors function by a dimerization mechanism with two receptors that recognize two independent HLA sites in the cell that the natural killer cell is investigating. Possibly, this dimerization is part of the activation or deactivation mechanism, depending on the class of receiver. In the case of GPVI, there may also be the possibility that two GPVI molecules associate with a Fcy, since each monomer of the Fcy dimer has a recognition sequence. But nevertheless,

10 stoichiometry is not yet known, and based on the structure of collagen, collagen-like peptides acting by GPVI and convulxin, it seems likely that the signal strength is related to the number of GPVI / Fcy complexes that are grouped between yes. Other platelet receptors that belong to that Ig family include ICAM-2 (CD) and PECAM (CD31).

All microorganisms, cell lines, expression systems, expression hosts, plasmids,

Promoters, resistance markers, origins of replication, restriction sites or other fragments or portions of vectors mentioned in the description not directly related to the claimed invention are generally available commercially or otherwise. Unless other allusions are given, they are used only as examples and are not essential with respect to the invention, and can be substituted by other suitable tools and biological materials, respectively.

The techniques that are essential according to the invention are described in detail below and above. Other techniques that are not described in detail correspond to known standard methods that are well known to a person skilled in the art, or are described in more detail in the cited references and patent applications and in the standard literature (for example Sambrook et al. , 1989, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor; Harlow, Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor).

25 EXAMPLES

Example 1: Materials - Protein A-Sepharose, goat anti-mouse and anti-rabbit antibodies conjugated with peroxidase, bovine serum albumin, Crotalus durissus terrificus venom, wheat germ agglutinin (WGA), agarose in pearls 4% cross-linked activated with N-hydroxylsuccinamidyl chloroformate, and Triton X-114 were from Sigma Chemical Co. (St Louis, MO), octanoyl-N-methyl-glucamide (ONMG) and nonanoyl-N-methyl-glucamide

30 (NNMG) were from Oxyl Chemie (Bobingen, Germany).

Example 2: Isolation of GPVI from platelets - Membrane glycoproteins were isolated from platelets as described above. Briefly, platelets (from 40 leucoplaquetary layers) were washed and lysed in 2% Triton X-114 in the presence of protease inhibitors. Triton X-114 and the aqueous phases were separated, and the detergent phase was loaded onto a wheat germ agglutinin column coupled to Sepharose 4B. The platelet glycoproteins were eluted with 10 mM Tris HCl, pH 7.4, 30 mM NaCl, 0.2% octanoyl-N-methylglucamide (ONMG) and 2% N-acetylglucosamine. After dialysis and concentration, the glycoprotein solution was loaded on a crosslinked agarose-bound convulxin column in crosslinked 4% beads activated with Nhydroxylsuccinamidyl-p-nitrophenyl chloroformate (1 mg / ml). The column was washed with 4 volumes of 10 mM Tris HCl, pH 7.4, 30 mM NaCl, 0.2% nonanoyl-N-methylglucamide (NNMG), and then with 4 volumes of 10 mM Tris HCl, pH 7, 4, NaCl 30

40 mM and 2% NNMG. GPVI was eluted with 0.08% SDS in 10 mM Tris / HCl, pH 7.4. The solution was concentrated and loaded onto a preparative 8.5% polyacrylamide gel using the Model 491 Prep Cell (BioRad, CA). Preparative electrophoresis was performed under unreduced conditions following the manufacturer's instructions. The GPVI eluted as a single band at 65 kDa. The fractions were pooled, concentrated in Centricon-30 (Amicon, Beverly, MA) and resuspended in 10 mM Tris / HCl, pH 7.4 and 0.1% ONMG.

Example 3: Amino acid analysis of GPVI-GPVI was digested with the LysC and AspN endoproteinases (Boehringer Mannheim, Germany). The 10 generated peptides were separated by reverse phase HPLC and sequenced in an Applied Biosystem model 477A pulsed liquid phase protein sequencer with a model 120A in-line phenylthiohydantoin amino acid analyzer.

Example 4: Amplification of a 221 bp fragment encoding part of GPVI from a cDNA library gt 50 77-

A sample (1010 pfu) (plaque forming units) was amplified from a human bone marrow library (Clonetech, Palo Alto, CA) using 2 combinations of 4 degenerate primers. The final primer concentrations were 2 µM, the dNTP concentration was 200 µM, and 2 U / 100 µl of AmpliTaq Gold (Perkin Elmer, Rotkreuz, Switzerland) reaction was used. The PCR conditions were 5 cycles at 37 ° C followed by 30 cycles at 44 ° C.

55 The 19mer sense 5 'TYATHCCNGCNATGAARMG 3' and the 20mer antisense 5 'TTRTANARNGCRAAYTGRTC 3' amplified a 221 bp fragment that was subcloned into Bluescript KS + (Stratagene, La Jolla, CA) and sequenced using the T7 Sequenase Switzerland kit (Amers Switzerland) ).

Claims (5)

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one.

Polyclonal anti-GPVI antiserum, anti-GPVI Fab obtainable from said anti-GPVI antiserum, or F (ab ') 2 anti-GPVI obtainable from said anti-GPVI antiserum, wherein GPVI has the sequence of SEQ ID NO: 3.

2.

Polyclonal anti-GPVI antiserum, anti-GPVI Fab obtainable from said anti-GPVI antiserum, or F (ab ’) 2 anti-GPVI

5 obtainable from said anti-GPVI antiserum according to claim 1, wherein GPVI can be obtained from platelets using wheat germ agglutinin and convulxin and applying gel electrophoresis on polyacrylamide of about 8.5%. 3. Polyclonal anti-GPVI antiserum, anti-GPVI Fab obtainable from said anti-GPVI antiserum, or F (ab ’) 2 obtainable from from said anti-GPVI antiserum according to claim 1 or 2, wherein the polyclonal anti-GPVI antiserum is generated in rabbits.

Four.

Fab fragment anti-GPVI of humanized mouse monoclonal antibodies against GPVI, in which GPVI has the sequence of SEQ ID NO: 3.

5.

Monoclonal or polyclonal antibodies that recognize the N-terminal region of GPVI, in which GPVI has the sequence of SEQ ID NO: 3.

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